Our goal is to develop effective protocols for systemic delivery of viral vectors to treat metastatic disease in immune competent hosts. In the funding period of grant CA RO1107082, we tested the hypothesis that cell carriers can carry viral vectors stealthily into tumors. We demonstrated that antigen specific CD8+T cells, pre-loaded with replication incompetent retrovirus, protect virus from complement and antibody and generate significant therapy (Nat Med 11:1073). Replacing the payload with a replication competent, oncolytic virus (VSV) generated significantly better therapy (Gen Ther 15:604). We also developed a novel approach in which naive T cells loaded with VSV can purge lymphoid organs of metastatic cells through oncolysis and priming of anti-tumor immunity (Nat Med 14:37). To address the critical issue of pre-existing immunity to the virus, we demonstrated that careful dosing of cyclophosphamide can modulate levels of neutralizing antibody to enhance systemic delivery (Clin Canc Res 14:259) and that the level with which the T cells are loaded with virus critically determines the sensitivity of loaded cells to pre-existing neutralizing antibody (Nat Med 14:37). These studies have led to completion of one trial to monitor trafficking of immune cells following intralymphatic delivery and two additional proposed clinical protocols. The overall hypothesis of this proposal, for renewal of Grant CA RO1107082, is that it will be possible to improve significantly on these results to generate effective therapy of well established metastatic tumors, resident in either the periphery and/or the lymphoid organs, using adoptive transfer of antigen specific, and/or antigen-non specific immune cells, pre-loaded with replication competent oncolytic virus. We have formulated 4 new Specific Aims: Aim 1: To increase anti tumor efficacy of adoptive T cell therapy with virus loaded, antigen specific lymphocytes by enhancing T cell trafficking, survival and activity using immune conditioning and alternative cell types as vehicles for enhanced T cell trafficking/virus delivery. Aim 2: To increase anti tumor efficacy of adoptive T cell therapy with virus loaded lymphocytes by enhancing the potency of the virus payload using oncolytic VSV expressing additional transgenes to increase T cell recruitment, direct cytotoxicity and/or immune activation. Aim 3: To enhance the efficiency with which oncolytic virus-loaded, antigen-non-specific cell carriers can purge metastatic tumors resident in lymphoid organs by increasing LN retention/trafficking and/or using alternative cell types to carry VSV to lymphoid organs. Aim 4: To use adoptive transfer of VSV-loaded cell carriers, in combination, to treat widespread metastatic disease in both naive, and virus-immune, mice using a model of large established s.c. tumors, LN and lung metastases arising from spontaneous metastasis from a primary B16.F10-ova tumor. Overall, the data from these experiments will guide the future development of our ongoing clinical trials, and the initiation of novel clinical trials for the use of VSV-loaded cell carriers as systemic agents at Mayo Clinic.